DNA (deoxyribonucleic acid)
DNA is the fundamental genetic material of all cells, and acts as a carrier of
genetic information.
 DNA is found;
 in the chromosomes in the nucleus and in the mitochondria in
eukaryotic organism, and
 in chloroplasts of plants.
 It consists of Oligonucleotides
Nucleic Acid Structure
 Nucleic acids are water soluble polymers of nucleotides.
 Nucleotides are composed of:
1. A pentose sugar, It is always ribose , (5 carbon sugar)
2. A purine or pyrimidine base,
3. A phosphate group
Bases:
The Purine Bases are:
Adenine (A)
Cytocine (C)
The pyrimidine bases are:
Thymine(T)
Guanione(G)
(Uracil)
Nucleotide
Dr. Fadwah Al-Ghalib
Molecular Biology
2nd Year
 The deoxyribonucleic acid, DNA, is a long chain of nucleotides
which consist of :
 Deoxyribose (a pentose = sugar with 5 carbons) indicating that it
lacks an oxygen molecule.
 Phosphoric Acid
 Organic (nitrogenous) bases (Purines - Adenine and Guanine, or
Pyrimidines -Cytosine and Thymine)
Double Strand DNA:
 The two strands are wound around each other into a double helix and
linked by hydrogen bonds between the bases of the nucleotides.
 The chain of nucleotides binds to complementary chain forming
double strand DNA, in the human genome.
 Adenine is always paired with a thymine
 Cytosine is always paired with a Guanine
The bases pairs are held together by hydrogen bonds:
 2-Bonds between A & T
 3-Bonds between C & G
 We can denature these bonds by the alteration of pH or by heating.
 Because there are three hydrogen bonds between G and C but only 2
between A and T, DNA that contains high concentrations of A and T
denatures at lower temperature than G and C rich DNA.
The Chromosome
 A chromosome is formed from a single DNA molecule that contains
many genes.
 DNA molecule is twisted onto itself, and the supercoiled molecule is
wrapped around proteins which maintain its shape. These proteins
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Dr. Fadwah Al-Ghalib
Molecular Biology
2nd Year
have a special name - histone proteins. The complex DNA protein is
called chromatin, while the structure formed by two turns of DNA
around one histone is called a nucleosome.
 A chromosomal DNA molecule contains three specific nucleotide
sequences which are required for replication:
 a DNA replication origin;
 a centromere to attach the DNA to the mitotic spindle.;
 a telomere located at each end of the linear chromosome.
 Human cells contain 46 chromosomes, whose total DNA is about one
to two meters in length.
 DNA is found associated with different types of proteins
(nucleoproteins), so if we want to study DNA we have to extract and
purify it first.
DNA Replication:
For DNA replication to occur the following are required:
 A DNA Template containing a region of single-stranded DNA
from which a complementary copy is made.
 Unwinding of DNA: The double-stranded helix must unwind,
and each strand then acts as a template.
 DNA helicase stimulates separation of two strands.
 The helix is unwound, assisted by the enzyme DNA gyrase. The
junction
Between the separated strands and the bound strands is called the
replication fork.
 Synthesis of RNA primers: RNA primers are produced by
primase which bind to the DNA. ( The primer is short strand of
RNA which is synthesized on the template at the start
replication and removed at the end.)
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Molecular Biology
2nd Year
 Elongation: Then DNA polymerase binds and begins to
synthesize DNA complementary to the parental strand.
 The direction of synthesis of DNA during replication, or RNA
during transcription is 5’→3’.
RNA Structure
I. RNA is single stranded
II. The sugar in RNA is ribose rather than deoxyribose
III. RNA has uracil (U) rather than T as one of its pyrimidines.
RNA exists in three forms:
1.
Messenger RNAs-are the nucleic acids that "record"
information from DNA in the cell nucleus and carry it to the
ribosomes and are known as messenger RNAs (mRNA).
2.
Transfer RNAs-The function of transfer RNAs (tRNA) is to
deliver amino acids one by one to protein chains growing at
ribosomes.
3.
Ribosomal RNAs-exist outside the nucleus in the cytoplasm
of a cell in structures called ribosomes. Ribosomes are small,
granular structures where protein synthesis takes place. facilitates the
interaction between mRNA and tRNA, resulting in the translation of
mRNA into protein.
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Dr. Fadwah Al-Ghalib
Molecular Biology
2nd Year
The genetic code: is read in groups of three nucleotides, each group
representing one amino acid, each trinucleotide sequence is called a
codon. (e.g, AGG (Arginine) GAG ( glycine), GUG (valine).
Gene expression:
Gene expression involves the transcription of a segment of DNA into
RNA, and the translation of RNA into a polypeptide.
 During gene expression information is retrieved from only one of
the two available strands.
 The segment of DNA containing a gene is first transcribed into a
single-stranded mRNA copy which has the same sequence of
bases as the sense strand of DNA, and is complementary to the
antisense strand.
 The DNA strand that is transcribed is called the template strand
(also known as the antisense strand), while its complement is
called the informational strand (also called the coding or sense
strand).
5' - G T A A T C C T C - 3' sense (partner)strand
|||||||||
3' - C A T T A G G A G - 5' antisense (template) strand
|||||||||
ppp 5' - G U A A U C C U C - 3'OH messenger RNA
-
Direction of transcription
 The sequence of bases is then translated into a sequence of amino
acids composing a polypeptide.
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Dr. Fadwah Al-Ghalib
Molecular Biology
2nd Year
Gene Transcription:
 The transcriptional unit of a gene is the region transcribed into a
primary into a primary RNA transcript, which is a precursor of
mRNA.
 It is made up of exons (containing expressed or coding DNA),
which are interrupted by sequences of unknown function known
as intervening sequences(IVS) or introns.
 Sequences on the 5’ side of a region of DNA are often called
“upstream”, whereas those on the 3’ side are “downstream”.
 The exon –intron complex is referred to as the open reading
frame.
 Cis-acting control elements influence gene transcription, by
acting as binding sites for protein produced by other genes, known
as trans-acting transcription factors or DNA-binding proteins.
They are located in the promoter and enhancer regions of the
gene.
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Dr. Fadwah Al-Ghalib
Molecular Biology
2nd Year
RNA splicing:
Process by which non-coding sequences of base pairs (introns) are subtracted from
the coding sequences (exons) of a gene in order to transcribe DNA into messenger
RNA (mRNA.).in other words (RNA splicing is a process that removes introns and
joins exons in a primary transcript).
Most introns start from the sequence GU and end with the sequence AG (in the 5' to
3' direction).
The general idea is shown in outline form in this figure
 In the cell nucleus, the DNA that includes all the exons and introns of the gene
is first transcribed into a complementary RNA copy called "nuclear RNA," or
nRNA.
 In a second step, introns are removed from nRNA by a process called RNA
splicing. The edited sequence is called "messenger RNA," or mRNA.
 During splicing all of the introns are usually removed, leaving all of the exons
in the mRNA. However, exons may also be removed during the splicing
process, resulting in variations in the final mRNA product and hence in the
polypeptide it encodes.
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Dr. Fadwah Al-Ghalib
Molecular Biology
2nd Year
 This process by which different mRNA transcripts are formed by removal of
different segments of the primary RNA transcript is known as Alternative
splicing.
Transcription:
Starts at the CAP site, so called because following transcription, the
5’end of the mRNA is capped at this site by the attachement of
aspecialized nucleotide (7-methyl-guanosine).
The CAP site is followed by a leader sequence leading up to the
initiation codon (ATG), that specifies the start of translation.
Transcription then proceeds, such that a full copy of the genes (introns
and exons) is made.
A stop codon (TAA, TAG,or TGA) indicates the end of the translated
region.
This is followed by a UTR (untranslated region) which includes the
poly(A) tail (polyadenylation).
Conclusion:
The newly synthezised RNA is modified by the following events:
 Capping—the addition of a nucleotide cap
 Polyadenylation—detachment of the RNA and addition of a string of a
adenosine residues.
 Splicing—sequences corresponding to introns are excised and discarded, and
the remaining exons are spliced together.
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Dr. Fadwah Al-Ghalib
Molecular Biology
2nd Year
Control Regions:
The Promoter region: is located immediately upstream of the genecoding region and contains sequence that govern the rate of transcription,
these include the TATA and CAAT boxes.
 TATA box: consists of an AT-rich sequence (often TATAA) which
occurs about 30 bp upstream from the transcriptional start site(often
denoted-30).
 CAAT box: Contains this short sequence about 80 bp upstream (-80)
of the start site.
 These sequences together with binding sites for other transcription
factors which vary according to the gene involved, are responsible for
the rate of transcription.
 The Initiator: is a sequence that is found in many promoters and defines
the startpoint of transcription
 The GC box is a common element in eukaryotic. Its consensus sequence
is GGGCGG. It may be present in one or more copies which can be located
between 40 and 100 bp upstream of the startpoint of transcription.
 Enhancers/Silencer regions: may be located within, near, or some
distance away
from the gene whose expression they stimulate or some
times suppress. They may provide an entry point for RNA polymerase or
they may bind other proteins that assist RNA polymerase to bind to the
promoter region
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Dr. Fadwah Al-Ghalib
Molecular Biology
2nd Year
Transcription is mostly controlled at the level of initiation
The binding of transcription factors to the promoter region of a gene attracts
RNA polymerases (RNA POL).
 There are three types of RNA POL ( I, II, III ).
 RNA I transcribes rRNA, it is found in the nucleolus.
 RNA POL II is located in the nucleoplasm (the part of the nucleus
excluding the nucleolus). Is responsible for synthesizing heterohenous
nuclear RNA (hnRNA), the precursor of mRNA.
 RNA III transcribes tRNA and other small RNAs.
 The promoters for RNA polymerase I and II are mostly upstream of
the startpoint, but some promoters for RNA polymerase III lie
downstream of the startpoint.
 There are three groups of factors that can act in conjunction with
RNA POL II:
a. The general factors: required for initiation of RNA synthesis
at all promoters, together they form a complex surroundings
the startpoint. The general factors together with RNA pol
constitute the basal transcription apparatus.
b. The upstream factors are DNA-binding protein, they increase
the efficiency of initiation and are required for the promoter to
function at an adequate level.
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Dr. Fadwah Al-Ghalib
Molecular Biology
2nd Year
c. The inducible factors: function in the same way as the
upstream factors but have a regulatory role. The are
synthezised or activated at specific times or in specific tissues.
The sequences that they bind are called response elements.
 The presence of proteins which act as transcription factors is required
before a RNA POL II molecule can recognize and bind to the
promoter of a gene and start transcription.
 These are referred to using the prefix TFII (e.g TFIIA, TFIIB, TFIID,
TFIIE,TFIIF). TFIID binds to the TATA box and is also known as
TATA factor.
 Binding of RNA POL II to the promoter region of a gene results in
localized separation of double-stranded DNA.
Mobile genetic elements
are present in all organisms. They are a major cause of spontaneous genetic
change and are now exploited as important tools for obtaining mutants,
isolating genes, and for studying gene expression.
DNA Recombination
DNA recombination refers to the phenomenon whereby two parental strands
of DNA are spliced together resulting in an exchange of portions of their
respective strands. This process leads to new molecules of DNA that contain
a mix of genetic information from each parental strand.
There are 3 main forms of genetic recombination. These are homologous
recombination, site-specific recombination and transposition.
Homologous recombination
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Dr. Fadwah Al-Ghalib
Molecular Biology
2nd Year
 Is the process of genetic exchange that occurs between any two
molecules of DNA that share a region (or regions) of homologous
DNA sequences.
 Indeed, it is the process of homologous recombination between the
maternal and paternal chromosomes that imparts genetic diversity to
an
organism.
Homologous
recombination
generally
involves
exchange of large regions of the chromosomes.
Site-specific recombination
 Involves exchange between much smaller regions of DNA sequence
(approximately 20 - 200 base pairs) and requires the recognition of
specific sequences by the proteins involved in the recombination
process.
 Site-specific recombination events occur primarily as a mechanism to
alter the program of genes expressed at specific stages of
development.
 The most significant site-specific recombinational events in humans
are the somatic cell gene rearrangements that take place in the
immunoglobulin genes during B-cell differentiation in response to
antigen presentation.
DNA Transposition
 Transposition is a unique form of recombination where mobile
genetic elements can virtually move from one region to another
within one chromosome or to another chromosome entirely.
 Transposition occurs with a higher frequency in bacteria and yeasts
than it does in humans.
 Is highly efficient means of generating recombinant DNA molecules
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Dr. Fadwah Al-Ghalib
Molecular Biology
2nd Year
 The exact nature of how transpositional events are controlled is
unclear.
 The identification of the occurrence of transposition in the human
genome resulted when it was found that certain processed genes were
present in the genome.
 These processed genes are nearly identical to the mRNA encoded by
the normal gene. The processed genes contain the poly(A) tail that
would have been present in the RNA and they lack the introns of the
normal gene.
 These particular forms of genes must have arisen through a reverse
transcription event, similar to the life cycle of retroviral genomes, and
then been incorporated into the genome by a transpositional event.
 Since most of the processed genes that have been identified are nonfunctional they have been termed pseudogenes.
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Dr. Fadwah Al-Ghalib
Molecular Biology
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